Cell type-specific repression of the immunoglobulin heavy chain enhancer

The murine immunoglobulin heavy chain (IgH) enhancer was studied as a model system for enhancer function and cell type-specific gene expression. Mutational analysis revealed that the IgH enhancer is a complex transcriptional element. The enhancer is comprised of a number of sequence motifs spread over a relatively large region ($\sim$400 bp) of DNA. While all of these sequence motifs are involved in modulation of enhancer activity, none of these sites is absolutely required for enhancer function as no single mutation completely abolishes activity of the full length enhancer. Deletion analysis suggested that the cell type-specificity of the IgH enhancer is due, in part, to negative control. Deletion of a small region of the enhancer leads to enhancer activation in nonlymphoid cells. Negative regulation of cell type-specificity was confirmed in experiments characterizing the transcriptional activity of oligonucleotides containing individual IgH enhancer protein binding sites. One protein binding site, $\mu$E5, functions as a positive transcription element in lymphoid cells and a negative transcription element in nonlymphoid cells. The cell type-specificity of the IgH enhancer was negated by overexpression of the $\mu$E5-binding transcription activator, ITF-1. Presumably, ITF-1 can derepress the enhancer by out competing the repressor for binding to the $\mu$E5 site. This result suggests that the cell type-specific regulation of the IgH enhancer can be mediated through the balance of positive and negative transcription factors rather than the absolute presence or absence of these regulatory proteins. In order to further expand this model of cell type-specific gene expression a cDNA encoding a protein capable of binding specifically to the $\mu$E5 site was isolated from a nonlymphoid cDNA library

Functional analysis of the murine IgH enhancer: evidence for negative control of cell-type specificity.

We have carried out a mutational analysis of the mouse IgH enhancer. Consistent with previous reports, deletions extending from either the 5' side or the 3' side of the enhancer fail to reveal distinct boundaries which define enhancer function in lymphoid cells. Interestingly, internal point mutations and deletions within the "enhancer core" regions fail to identify any necessary functional role for these conserved elements. When tested in CV1 cells, which do not normally respond to the IgH enhancer, certain deletions exhibit significant enhancer activity. We take these findings to indicate that the functional domains of the IgH enhancer are complex and that cell type specificity is defined in part by negative factors present in non-lymphoid cells

Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer.

The activity of the immunoglobulin heavy-chain (IgH) enhancer is restricted to B cells, although it binds both B-cell-restricted and ubiquitous transcription factors. Activation of the enhancer in non-B cells upon overexpression of the basic helix-loop-helix (bHLH) protein E2A appears to be mediated not only by the binding of E2A to its cognate E box but also by the resulting displacement of a repressor from that same site. We have identified a "two-handed" zinc finger protein, denoted ZEB, the DNA-binding specificity of which mimics that of the cellular repressor. By employing a derivative E box that binds ZEB but not E2A, we have shown that the repressor is active in B cells and the IgH enhancer is silenced in the absence of binding competition by bHLH proteins. Hence, we propose that a necessary prerequisite of enhancer activity is the B-cell-specific displacement of a ZEB-like repressor by bHLH proteins.</jats:p

Fusion FISH Imaging: Single-Molecule Detection of Gene Fusion Transcripts <i>In Situ</i>

<div><p>Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the <i>in situ</i> detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence <i>in situ</i> hybridization (sm-FISH), in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing's sarcoma cells. This technology should pave the way for accurate <i>in situ</i> typing of many cancers that are associated with, or caused by, fusion transcripts.</p> </div